Membrane-targeting antimicrobials as promising resistance-breaking antibiotic candidates

Abstract

Antimicrobial resistance has rapidly become one of the biggest threats to global health; a crisis only exacerbated by a lack of new antibiotic development. Currently, the bacterial cell envelope is a primary target of several antibiotics. This structure differs between Gram-positive and Gram-negative organisms; the most striking variation being the presence of an additional Gram-negative outer membrane. However, developing antimicrobial agents with similar modes of action to current cell envelopetargeting antimicrobials such as β-lactams and vancomycin may remain susceptible to pre-existing bacterial resistance mechanisms. Therefore, there is the urgent need for agents acting on thus far unexploited targets. Two such emerging strategies are targeting of the bacterial cytoplasmic membrane in both Gram-positive and Gram-negative bacteria and disrupting the outer membrane in Gram-negative bacteria to improve efficacy of already approved antibiotics. Therefore, the aim of this thesis was to develop multiple fluorescence-based assays to screen for these effects in both novel natural product compounds and current clinically used antibiotics. Through this, I was able to develop fluorescence-based assays for the detection of both inner membrane depolarisation and permeabilisation in the Gram-positive and Gram-negative model organisms, Bacillus subtilis and Escherichia coli respectively, and techniques investigating outer membrane permeabilisation and multidrug efflux inhibition in E. coli. Implementation of these screens allowed for the identification of several Actinomycete-derived antimicrobial extracts with cytoplasmic membrane effects in B. subtilis. Preliminary mode of action studies of these were also performed using further microscopic experimentation. Finally, I utilised these techniques to further elucidate the mode of action of two clinically relevant membrane-targeting antimicrobials: daptomycin and octenidine, and investigate the membrane effects of PanT toxins identified from various bacteria and bacteriophages.